Method for diagnosing irritable bowel syndrome and monitoring inflammatory bowel disease

ABSTRACT

A method for aiding in differentiating irritable bowel syndrome from inflammatory bowel disease by determining the level of total endogenous human lactoferrin in clinical specimens, such as feces, mucus and bile, wherein an elevated level of lactoferrin substantially precludes diagnoses of IBS and other noninflammatory etiologies, and a kit usable in such method are provided. Further provided is a method for quantitating the level of total endogenous human lactoferrin in clinical specimens, such as feces, mucus and bile, to monitor gastrointestinal inflammation in persons having inflammatory bowel disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 10/629,975, which is a divisional of U.S.application Ser. No. 10/002,842 filed on Nov. 14, 2001, which claims thebenefit of priority to U.S. Provisional Application Ser. No. 60/248,288,filed on Nov. 14, 2000, each of which is hereby incorporated byreference.

BACKGROUND

The present invention relates to the clinical differentiation andmonitoring of gastrointestinal illnesses. More particularly, the presentinvention relates to a method for aiding in differentiating irritablebowel syndrome from inflammatory bowel disease by determining the levelof total endogenous human lactoferrin in clinical specimens, such asfeces, mucus and bile, wherein an elevated level of lactoferrinsubstantially precludes diagnoses of IBS and other noninflammatoryetiologies, and a kit usable in such method. The present inventionfurther relates to a method for quantitating the level of totalendogenous human lactoferrin in clinical specimens, such as feces, mucusand bile, to monitor gastrointestinal inflammation in persons havinginflammatory bowel disease.

Gastrointestinal illnesses are responsible for an extensive loss of lifeworldwide. For instance, diarrhea is a major cause of morbidity andmortality in developing countries with an estimated one billion cases ofdiarrheal diseases and five million deaths in children per year. In theUnited States, eight to twelve million people are treated each year forinfectious diarrhea making up 2.5% of total hospitalizations andresulting in 10,000 deaths. Other gastrointestinal illnesses includeinflammatory bowel disease (IBD) and irritable bowel syndrome (IBS).Annual evaluation for these disorders in the United States results in 1and 3.5 physician visits, respectively. Symptoms of active IBS and thoseof active IBD are similar and, accordingly, the two diseases oftenpresent nearly identically. However, IBD can be a severe,life-threatening condition and thus quick, accurate differentialdiagnosis is extremely important.

IBD, comprised of both Crohn's Disease (CD) and ulcerative colitis (UC),is characterized by a chronic immune-mediated inflammatory response thatresults in histologic damage to the intestinal lining. Both CD and UCexhibit large numbers of leukocytes that migrate to the mucosa and intothe intestinal lumen. Both diseases oscillate between active (i.e.,presence of intestinal inflammation) and inactive (i.e., minimal to nointestinal inflammation) stages of disease activity. Active IBD caninclude symptoms such as bloody diarrhea, abdominal pain, and fever. Theinactive stage has minimal to no intestinal inflammation and lackssevere gastrointestinal illness.

Patients who have active IBD but who exhibit mild signs and symptoms maybe difficult to distinguish from patients with active IBS, an intestinaldisorder of motility and the intestinal nervous system. Unlike IBD, IBSdoes not involve intestinal inflammation. In persons with IBS, theintestine appears normal upon endoscopic examination and leukocytes arenot present in the mucosa or in fecal specimens. Symptoms can mimicthose of IBD and include bloating, diarrhea, constipation, and severeand often debilitating abdominal pain. It is estimated that at least 35million Americans suffer from IBS.

The similarity in symptoms between IBS and IBD renders rapid diagnosisrather difficult. However, given the potential severity of untreatedIBD, differential diagnosis is crucial. The diagnosis ofgastrointestinal illnesses, in general, is aided by diagnostic testssuch as enzyme-linked immunosorbant assays (ELISAs), latex agglutinationand lateral flow immunoassay. These tests are rapid and inexpensivemethods for detecting markers in feces for enteric pathogens andinflammation. One marker of particular interest that has been found tobe most specific for leukocytes in fecal specimens is lactoferrin. Humanlactoferrin is an 80 kilodalton glycoprotein. This iron-binding proteinis secreted by most mucosal membranes. It is a major component of thesecondary granules found in polymorphonuclear neutrophils (PMNs), aprimary component of the acute inflammatory response. Otherhematopoietic cells such as monocytes and lymphocytes, do not containlactoferrin, whereas various bodily secretions contain levels in themg/mL range. During the process of inflammation, PMNs infiltrate themucosa lining of the small and large intestine. This increase in thenumber of activated tissue leukocytes and exudation of plasma fromulcerated mucosa results in an increase in the level of lactoferrinfound in feces. The protein is resistant to proteolysis and, as such, itprovides a useful non-invasive fecal marker of intestinal inflammation.

Human lactoferrin has been used as a marker for fecal leukocytes in anumber of applications. For instance, fecal lactoferrin has been used asa marker for leukocytes to distinguish noninflammatory diarrhea frominflammatory diarrhea, as disclosed in U.S. Pat. No. 5,124,252 (the “252patent”). Noninflammatory diarrhea caused by agents such as rotavirus,Norwalk-like agents and cholera, typically causes minimal to nointestinal damage and patients respond readily to oral rehydration.Inflammatory diarrheas include those caused by enteric pathogens such asClostridium difficile, Shigella species, Salmonella species,Campylobacter jejuni and Entamoeba histolytica and those that have noclearly defined infectious agent such as CD and UC. U.S. Pat. No.5,124,252 discloses an in vitro test for fecal leukocytes which aids indistinguishing inflammatory from noninflammatory diarrhea. The >252patent discloses testing fecal samples suspected of containingleukocytes with an assay that utilizes an antibody for lactoferrin todetermine the presence of leukocytes in the fecal sample.

Human lactoferrin also has been used as a marker for diagnosis ofinflammatory gastrointestinal disorders, colon polyp and colorectalcancer as disclosed in U.S. Pat. No. 5,552,292 (the “292 patent”).However, neither the method of the '252 patent nor that of the '292patent disclose utility in distinguishing IBS and IBD. The samplestested by the assay of the '252 patent are samples suspected ofcontaining leukocytes. This suspicion is owed to the patient presentingwith diarrhea. However, 25-50% of persons having IBD do not present withdiarrhea and, thus, the '252 patent does not relate to diagnosingetiology in such patients. As for the '292 patent, the disclosed methodutilizes a 1:100 sample dilution which does not allow for accuratequantitation of lactoferrin levels. Further, the '292 patent disclosesusing partial forms of molecules for testing and not total endogenouslactoferrin, again affecting the accuracy of the quantitation. Themethod of the '292 patent also does not relate to utilizing lactoferrinlevels to distinguish between IBD and IBS. The population tested in the'292 patent, while including persons with UC and CD, did not includepersons having IBS. Therefore, there remains a need in the diagnosticindustry for a noninvasive method for differentially diagnosing IBD andIBS which utilizes human lactoferrin as a marker.

Given that lactoferrin has been shown to be a good marker for fecalleukocytes, tests have been developed to aid physicians in determiningthe presence of fecal lactoferrin. One such test is the LEUKO-TEST®,manufactured by TechLab, Inc. of Blacksburg, Va. The LEUKO-TEST® is alatex agglutination test for detecting fecal lactoferrin. It isnoninvasive and demonstrates active intestinal inflammation thusproviding physicians evaluating patients with diarrhea with importantinformation concerning the severity of any underlying bacterialinfection.

Even though the LEUKO-TEST® is useful for evaluating gastrointestinalillnesses, the latex agglutination format provides some limitations. Inlarge hospitals with a high volume of specimens, batching is preferred.A format such as ELISA is more useful for batching than latexagglutination and has the option of automation. It also may indicateseverity of the disease and the effectiveness of medical treatments, bymeasuring the levels of fecal lactoferrin. In the case of IBD, a rise infecal lactoferrin may provide an early indicator for active disease andthe effects of medical treatments.

Currently, there are no known in vitro diagnostic aids to assisttreating physicians, or other clinical personnel, in distinguishingbetween IBD and IBS. Accordingly, there remains a need for an in vitrodiagnostic aid to assist treating physicians and other clinicalpersonnel in distinguishing between these two commonly presentingdiseases.

SUMMARY

Accordingly, the present invention provides a non-invasive method fordifferentiating irritable bowel syndrome (IBS) from inflammatory boweldisease (IBD) wherein the presence of fecal lactoferrin is used as adetection marker for fecal leukocytes, elevated levels of whichsubstantially preclude diagnoses of IBS and other noninflammatoryetiologies, and a kit therefor. This rapid diagnosis then may beutilized by healthcare professionals to prescribe proper treatment. Thepresent invention further provides immunoassays, e.g., enzyme-linkedimmunoassays (ELISAs), that utilize antibodies specific to humanlactoferrin for the measurement of total endogenous lactoferrin inclinical specimens, such as human feces, mucus and bile, and a kitusable in such immunoassays. Still further, the present inventionprovides to a method for quantitating the levels of lactoferrin fromendogenous sources, particularly, infiltrating leukocytes, to monitorgastrointestinal inflammation in persons having IBD.

It has been shown that fecal lactoferrin has utility as a marker fordistinguishing patients with IBD from those with less severe IBS. To aidphysicians and other clinical personnel in utilizing this discovery,immunoassays are provided herein for detecting elevated levels of fecallactoferrin and for quantitating fecal lactoferrin levels. Specifically,a qualitative enzyme-linked immunosorbent assay (ELISA) is providedwherein polyclonal antibodies against total endogenous human lactoferrinare utilized to detect elevated levels of fecal lactoferrin. Thequalitative assay of the present invention permits the screening ofpatients presenting with symptoms common between IBS and IBD. Ifelevated levels of fecal lactoferrin are detected, a diagnosis of IBS issubstantially precluded. It will be understood and appreciated by thoseof skill in the art that a qualitative immunoassay such as a lateralflow dipstick that utilizes both monoclonal and polyclonal antibodies tototal endogenous lactoferrin also may be used to indicate the absence orpresence of gastrointestinal inflammation. Such is contemplated to bewithin the scope hereof.

The qualitative assay of the present invention provides a test that iseasy to use, simple to read, and accurate for distinguishing active IBDfrom active IBS. To substantiate equivalence of the ELISA to predicatedevices, test results have been compared to microscopy results and toresults from the latex agglutination test manufactured by TechLab, Inc.of Blacksburg, Va. under the brand name LEUKO-TEST®. To this end, twostudies were conducted involving a total of 166 fecal specimens. Whencompared with microscopy, the assay of the present invention presented asensitivity of 80.0% in the first study and 94.1% in the second study.The assay further presented a specificity of 90.0% in the first studyand 51.7% in the second study. In the same studies, when compared withthe LEUKO-TEST®, sensitivity results were 90.5% in the first study and89.6% in the second study. Specificity results were 86.4% in the firststudy and 57.5% in the second study.

For the evaluation of the qualitative assay of the present invention asa diagnostic aid for IBD and IBS patients, fecal samples from subjectshaving IBD were collected and the assay results were compared with thosefrom healthy control subjects and subjects having clinically definedcases of IBS. The IBD group included subjects having both ulcerativecolitis (UC) and Crohn's disease (CD). The fecal lactoferrin levelsdetermined in these subjects were used to establish the preferredpredictive optical density for the assay of 0.200 OD₄₅₀. Resultsindicated that the assay was positive (i.e., an OD₄₅₀ greater than orequal to 0.200) for 86.0% of fecal specimens from subjects with activeIBD and was consistently negative (i.e., an OD₄₅₀ less than 0.200) forspecimens from subjects with active IBS and from healthy controlsubjects. (“OD₄₅₀” as used herein indicates an optical density measuredat 450 nm on a single wavelength spectrophotometer.)

In an additional clinical evaluation the qualitative assay of thepresent invention was compared to clinical assessments of IBD and activeIBS subjects. In the IBD group, there were ninety-two subjects withactive disease (fifty-one with active CD and forty-one with active UC)and fifty-seven with inactive disease. In the active group, a total ofeighty subjects, or 87.0%, tested positive with the assay of the presentinvention. In the inactive group, thirty-two, or 56.1%, tested positive.Of the fifty-one IBD subjects with active CD, forty-four, or 86.3%,tested positive. Of the forty-one IBD subjects with active UC,thirty-six, or 87.8%, tested positive with the assay. All thirty-onesubjects, or 100%, with active IBS, and all fifty-six healthy controlsubjects, or 100%, tested negative with the assay of the presentinvention.

Research findings thus support the use of the qualitative assay of thepresent invention as an in vitro diagnostic aid for detecting elevatedlevels of lactoferrin as a detection marker for fecal leukocytes and anindicator of inflammation. Other intestinal ailments, including manygastrointestinal infections and colorectal cancer, often result inelevated levels of lactoferrin in fecal specimens and these specimenslikely will test positive with the assay of the present invention.Therefore, a diagnosis of active IBD cannot be established solely on thebasis of a positive result with the assay of the present invention.However, a positive result with the assay of the present invention willpermit the substantial preclusion of a diagnosis of IBS or othernoninflammatory etiologies.

Also provided is a quantitative ELISA wherein polyclonal antibodiesagainst total endogenous human lactoferrin are utilized to quantitatelevels of gastrointestinal inflammation through comparison to a standardcurve generated using purified human lactoferrin. These levels then maybe utilized to monitor the effects of medical treatments in patientshaving IBD.

In the quantitative assay of the present invention, the level of totalendogenous human lactoferrin in clinical specimens is determined throughcomparison to a standard curve generated using purified humanlactoferrin and analyzed by linear regression. Research findings showthat the level of fecal lactoferrin in persons having IBS was lower thanthe mean fecal lactoferrin level determined in healthy personsindicating the absence of gastrointestinal inflammation. However, thelevels of fecal lactoferrin in IBD patients determined using thequantitative assay of the present invention were significantly higherthan the mean fecal lactoferrin level of healthy persons. Thus, thequantitative assay of the present invention will permit the monitoringof patients having IBD as the levels of fecal lactoferrin may bedetermined over the course of medical treatments to determine whether ornot the treatment is effective in decreasing or eliminatinggastrointestinal inflammation.

Additional aspects of the invention, together with the advantages andnovel features appurtenant thereto, will be set forth in part in thedescription which follows, and in part will become apparent to thoseskilled in the art upon examination of the following, or may be learnedfrom the practice of the invention. The aspects and advantages of theinvention may be realized and attained by means, instrumentalities andcombinations particularly pointed out in the appended claims.

DETAILED DESCRIPTION

The present invention is directed to diagnostic test methods for aidingin differentiating irritable bowel syndrome (IBS) from inflammatorybowel disease (IBD) and for monitoring persons having IBD, and a kitusable in such methods. The particular embodiments described herein areintended in all respects to be illustrative rather than restrictive.Alternative embodiments will become apparent to those skilled in the artto which the invention pertains without departing from its scope.

The qualitative diagnostic test method of the present invention is animmunoassay for the detection of elevated levels of lactoferrin, adetection marker for fecal leukocytes, and an indicator of intestinalinflammation. The method can be used as an in vitro diagnostic aid tohelp identify patients with active IBD and rule out those with activeIBS, which is noninflammatory. The lactoferrin specific immunoassays canbe used to differentiate IBS from IBD by measuring the level of totalendogenous lactoferrin. “Total endogenous lactoferrin,” as that term isused herein, comprises lactoferrin derived from endogenous sources,particularly infiltrating leukocytes (i.e., leukocytes, plasma, bile andmucosal secretions).

In the preferred embodiment, the qualitative immunoassay of the presentinvention is an enzyme-linked immunoassay (ELISA). The ELISA formatprovides the clinical laboratory with a simple-to-use test that isfamiliar to medical and clinical laboratory personnel. The test will aida treating physician and other clinical personnel in distinguishingactive IBD, which can become life-threatening and requires specialtreatment, from IBS, which is not life-threatening and which utilizeslifestyle modifications as therapy. The test is easy to perform,utilizing a one component substrate system and a total incubation timeof seventy-five minutes. The qualitative assay of the present inventionpreferably utilizes a specimen dilution of 1:400 and optical densitiesof 0.200 OD₄₅₀ and 0.160 at OD_(450/620). It will be understood andappreciated by those of skill in the art that a qualitative immunoassaysuch as a lateral flow dipstick that utilizes both monoclonal andpolyclonal antibodies to total endogenous lactoferrin also may be usedto indicate the absence or presence of gastrointestinal inflammation.Such is contemplated to be within the scope hereof.

The following are examples of procedures which have been utilized toestablish the preferred qualitative and quantitative assays according tothe present invention. The following examples are merely exemplary andnot presented by way of limitation.

1. Qualitative Assay a. Establishment of Optimal Sample Dilution Factorand Optical Density

The assay of the present invention was designed and developed to detectlevels of fecal lactoferrin at a lower level detectable by predicatedevices, specifically the LEUKO-TEST®. The lower limit of detection ofthe LEUKO-TEST® is 256 ng/mL with purified human lactoferrin. In theLEUKO-TEST®, a specimen dilution of 1:50 and a minimum limit ofdetection of 256 ng/mL provides a lower limit of detection in fecalspecimens of approximately 12 μg/mL. A specimen dilution of 1:400 and aminimum detection limit for the assay of the present invention of 32ng/mL also provides a lower limit of detection in fecal specimens ofapproximately 12 μg/mL. Accordingly, a 1:400 specimen dilution waschosen for the assay of the present invention. Similarly, an opticaldensity of 0.200 OD₄₅₀ for the assay was chosen. (As used herein, OD₄₅₀indicates an optical density obtained spectrophotometrically at 450 nmon a single wavelength spectrophotometer.)

It will be understood and appreciated by those of skill in the art thatthe preferred dilution factor and optical densities have been determinedbased upon reagents currently available and deemed to be optimal.However, reagents other than those now desired may become improved anddesirable over time. Variations in reagents may producepreferable/optimal dilution factors and/or optical densities other thanthose determined herein. Such variations are contemplated to be withinthe scope of the present invention. The key to determining optimalvalues is based upon sensitivity as more fully described below.

To verify that the 1:400 specimen dilution provides the most desirablesensitivity with the current reagents, 121 fecal specimens were analyzedcomparing a 1:400 dilution to a 1:800 dilution. (Sensitivity iscalculated herein by dividing the number of samples taken from subjectswith IBD which produce a positive result in the assay by the number ofsamples taken from subjects with IBD.) Test results additionally wereevaluated comparing OD₄₅₀ values of 0.200 to OD₄₅₀ values of 0.300.Results were compared with microscopy for fecal leukocytes and with theLEUKO-TEST®. The results are summarized in Tables I-VIII below.

TABLE I Comparison of the ELISA with microscopy for fecal leukocytesusing a 1:400 dilution and an OD450 of 0.200 ELISA vs. microscopy (N =121) Microscopy positive Microscopy negative ELISA positive 32 42 ELISAnegative 2 45 Relative Sensitivity 94.0% Relative Specificity 52.0%Correlation 64.0%

TABLE II Comparison of the ELISA with microscopy for fecal leukocytesusing a 1:400 dilution and an OD450 of 0.300 ELISA vs. microscopy (N =121) Microscopy positive Microscopy negative ELISA positive 31 31 ELISAnegative 3 56 Relative Sensitivity 91.0% Relative Specificity 64.0%Correlation 72.0%

TABLE III Comparison of the ELISA with microscopy for fecal leukocytesusing a 1:800 dilution and an OD450 of 0.200 ELISA vs. microscopy (N =121) Microscopy positive Microscopy negative ELISA positive 30 31 ELISAnegative 4 56 Relative Sensitivity 88.0% Relative Specificity 64.0%Correlation 77.0%

TABLE IV Comparison of the ELISA with microscopy for fecal leukocytesusing a 1:800 dilution and an OD450 of 0.300 ELISA vs. microscopy (N =121) Microscopy positive Microscopy negative ELISA positive 26 24 ELISAnegative 8 63 Relative Sensitivity 77.0% Relative Specificity 72.0%Correlation 74.0%

TABLE V Comparison of the ELISA with the LEUKO-TEST ® using a 1:400dilution and an OD450 of 0.200 ELISA vs. LEUKO- TEST ® LEUKO-TEST ®LEUKO-TEST ® (N = 121) positive negative ELISA positive 43 31 ELISAnegative 5 42 Relative Sensitivity 89.6% Relative Specificity 57.5%Correlation 70.2%

TABLE VI Comparison of the ELISA with the LEUKO-TEST ® using a 1:400dilution and an OD450 of 0.300 ELISA vs. LEUKO- LEUKO-TEST ®LEUKO-TEST ® TEST ® (N = 121) positive negative ELISA positive 41 21ELISA negative 7 52 Relative Sensitivity 85.0% Relative Specificity71.2% Correlation 77.0%

TABLE VII Comparison of the ELISA with the LEUKO-TEST ® using a 1:800dilution and an OD450 of 0.200 ELISA vs. LEUKO- LEUKO-TEST ®LEUKO-TEST ® TEST ® (N = 121) positive negative ELISA positive 39 22ELISA negative 9 51 Relative Sensitivity 81.3% Relative Specificity69.9% Correlation 74.4%

TABLE VIII Comparison of the ELISA with the LEUKO-TEST ® using a 1:800dilution and an OD450 of 0.300 ELISA vs. LEUKO- LEUKO-TEST ®LEUKO-TEST ® TEST ® (N = 121) positive negative ELISA positive 34 16ELISA negative 14 57 Relative Sensitivity 70.8% Relative Specificity78.1% Correlation 75.2%

In summary, a fecal specimen dilution of 1:400 and an assay OD₄₅₀ of0.200 showed the highest level of sensitivity with the current reagents.Accordingly, these conditions were determined to be optimal for theassay of the present invention. Normal fecal specimens contain lowlevels of lactoferrin and the 1:400 dilutions have been determined to beoptimal in detecting an increase in lactoferrin over background levels.The use of dilutions lower than 1:400 may result in positive testresults due to the presence of normal lactoferrin levels.

b. Collection of Specimens and Preparation of Dilutions

Standard collection and handling procedures typically used for fecalspecimens for culture may be used in collecting samples for the assay ofthe present invention. In the preferred embodiment, fecal specimens areto be tested within twenty-four hours of collection. However, if theassay is not to be performed within forty-eight hours of collection, itis preferred that the specimens be stored at −20° C. or lower.Additionally, it is preferred that collected specimens be transportedand diluted in the Diluent as soon as possible after collection and,once diluted, that the specimens be stored at between about 2° C. andabout 8° C. It is preferred that the specimens be mixed (i.e., using avortex mixer) thoroughly prior to performing the assay of the presentinvention. This includes complete mixing of the specimen prior totransfer to the Diluent, as more fully described below, as well ascomplete mixing of the diluted specimen prior to performing the assay.

The following method was used to prepare a diluted specimen from aliquid fecal specimen. Two plastic tubes were set up for each specimento be tested. For each specimen, 950 μL of 1× Diluent (prepared as morefully described below) subsequently was added to each of the two tubes.Using a transfer pipette, one drop (i.e., approximately 50 μL) of liquidfecal specimen was added to one of the tubes and thoroughly mixed usinga vortex mixer. Subsequently, one drop of the diluted specimen wastransferred into the second tube containing 950 μL of 1× Diluent(prepared as more fully described below). The result was a 1:400dilution of the specimen in the second tube. Thus, only the second tubewas used for the remainder of the test procedure.

The following method was used to prepare a diluted specimen from aformed or solid fecal specimen. Two plastic tubes were set up for eachspecimen to be tested. For each specimen, 1.9 mL of 1× Diluent (preparedas more fully described below) was added to only one of the two tubes.Subsequently, 0.10 g of fecal specimen were added to this tube (1:10)and thoroughly mixed using a vortex mixer. Next, 950 μL of the 1×Diluent (prepared as more fully described below) was added to the secondtube and one drop (i.e., approximately 50 μL) of the previously dilutedspecimen is transferred into the second tube. The result was a 1:400dilution of the specimen in the second tube. Thus, only the second tubewas used for the remainder of the test procedure.

The specimen in the second tube prepared according to either of theabove procedures was mixed in a vortex mixer for approximately tenseconds and subsequently stored at between about 2° C. and about 8° C.until the remainder of the test procedure was performed. Prior totransferring the diluted specimen into a microtiter well according tothe test procedure, as more fully described below, the specimen wasthoroughly mixed in the vortex mixer once again. This procedure soughtto ensure thorough mixing of the specimen.

c. Necessary Test Reagents and Preparation Thereof

A number of reagents were necessary to carry out the preferredembodiment of the qualitative assay of the present invention. Thesereagents included 10× Diluent, 1× Diluent, Conjugate, Substrate,Positive Control, Wash Buffer Solution and Stop Solution. The 10×Diluent was a 10× concentrate of buffered protein solution containing0.2% thimerosal as a preservative. The Diluent was supplied as a 10×concentrate. Therefore, to prepare the 1× Diluent necessary for theassay of the present invention, a total volume of 400 mL was diluted byadding 40 mL of the 10× concentrate to 360 mL of deionized water. Anyunused 1× Diluent was stored at between about 2° C. and about 8° C.

The Conjugate used with the assay of the present invention preferablycomprises rabbit polyclonal antibody specific for human lactoferrinconjugated to horseradish peroxidase and in a buffered protein solutioncontaining 0.02% thimerosal as a preservative. The Substrate used withthe assay of the present invention preferably comprises a solutioncontaining tetra-methyl-benzidine substrate and peroxidase. The PositiveControl used with the assay of the present invention preferablycomprises human lactoferrin in a buffered protein solution containing0.02% thimerosal as a preservative. The Stop Solution used with theassay of the present invention preferably comprises 0.6 N sulfuric acid.

The Wash Buffer Solution used with the assay of the present inventionwas supplied as a 20× concentrate containing phosphate buffered saline,detergent and 0.2% thimerosal as a preservative. To prepare the 1× WashSolution necessary for the assay of the present invention, a totalvolume of one liter of concentrate was diluted by adding 50 mL of theconcentrate to 950 mL of deionized water. Any unused 1× Wash Solutionwas stored at between about 2° C. and about 8° C.

Microassay plates containing twelve strips and eight wells per strip arepreferred for the assay of the present invention. Each specimen and eachcontrol requires a single coated well. To prepare the plates, each stripwas coated with purified polyclonal antibody specific for lactoferrin.Microassay plates were stored with desiccant.

All reagents were stored at room temperature prior to use in the assayof the present invention.

The present invention includes a kit designed and prepared for carryingout the quantitative assay. In the preferred embodiment, the kitcontains 40 mL 10× Diluent, 7 mL Conjugate, 14 mL Substrate, 3.5 mLPositive Control, 50 mL Wash Buffer Solution, 7 mL Stop Solution and onemicroassay plate stored with desiccant. The assay of the presentinvention utilizes antibodies to human lactoferrin. The microassay platesupplied with the kit contains immobilized polyclonal antibody againstlactoferrin. The detecting antibody consists of polyclonal antibodyconjugated to horseradish peroxidase.

d. Test Procedure

To perform the qualitative assay of the present invention, initially thenumber of wells needed was determined. Each specimen or control requiredone well and, therefore, the number of wells was determined accordingly.Next, one drop (i.e., about 50 μL) of Positive Control was added to asingle well designated the Positive Control Well and one drop (i.e.,about 50 μL) of 1× Diluent was added to a single well designated theNegative Control Well. Subsequently, two drops (i.e., about 100 μL) of1:400 diluted specimen (prepared according to the above procedure) wasadded to a third well and all wells were incubated at about 37EC (“2°C.) for approximately thirty minutes. After incubation, the contents ofthe assay wells was discarded into a discard pan.

Next, each well was washed using 1× Wash Solution (prepared as describedabove) and placed in a squirt bottle with a fine-tipped nozzle. In thismanner, the 1× Wash Solution was directed into the bottom of each of thewells with some force. Each well was filled with the 1× Wash Solutionand the contents thereof subsequently discarded into a discard pan. Themicroassay plate was then inverted and slapped on a dry paper towel.This wash procedure was performed a minimum of four times using a drypaper towel each time. If any particulate matter was observed in thewells, the washing procedure was continued until all the matter wasremoved.

Subsequently, one drop (i.e., about 50 μL) of Conjugate was added toeach well and the wells were incubated at about 37° C. (±2° C.) forapproximately thirty minutes. After incubation, the contents of theassay wells were discarded into a discard pan and the washing procedurewas repeated. Next, two drops (i.e., about 100 μL) of Substrate wereadded to each well and the wells were gently tapped to mix the contents.The wells were then incubated at room temperature for approximatelyfifteen minutes. The wells were gently tapped a couple of times duringthe incubation period.

Next, one drop (i.e., 50 μL) of Stop Solution was added to each well andthe wells were gently tapped. The wells were allowed to sit at roomtemperature for about two minutes before reading. The addition of StopSolution converted the blue color to a yellow color which could then bequantified by measuring the optical density at 450 nm on a microplateELISA reader. The instrument was blanked against the negative controland the underside of each well was wiped before measuring the opticaldensity. Optical densities (OD₄₅₀ and OD_(450/620)) were recorded forthe Positive Control Well, the Negative Control Well and each specimentested. (“OD_(450/620)” as used herein indicates an optical densityobtained spectrophotometrically at 450/620 nm on a dual wavelengthspectrophotometer.) Readings of duplicate wells were averaged before theresults were interpreted.

The specified test procedure represents the preferred embodiment asoptimal results are obtained by following the procedure specifiedbecause the reagents, concentrations, incubation conditions, andprocessing specifications have been optimized for sensitivity andspecificity. Accordingly, alterations of the specified procedure and/orof the indicated test conditions may affect the sensitivity andspecificity of the test.

e. Quality Control

The positive and negative control must meet certain criteria for thetest to be valid. First of all, the Positive Control Well must be avisible yellow color and, when read on a spectrophotometer, it must havean OD₄₅₀ and OD_(450/620)>0.500. The Negative Control Well must have anOD₄₅₀<0.200 or an OD_(450/620)<0.160. To ensure that carryover has notoccurred, testing should be repeated if a sample gives a weak positiveresult (i.e, <0.400) and is adjacent to a strong positive well.

f. Interpretation of Results

Optical densities were measured at 450 nm on a single wavelengthspectrophotometer and at 450/620 nm on a dual wavelengthspectrophotometer. On a single wavelength spectrophotometer, an OD₄₅₀ ofless than 0.200 indicated a negative result and an OD₄₅₀ of greater thanor equal to 0.200 indicated a positive result. On a dual wavelengthspectrophotometer, an OD_(450/620) of less than 0.160 indicated anegative result and an OD_(450/620) of greater than or equal to 0.160indicated a positive result.

A positive test result indicated the specimen contained elevated levelsof lactoferrin when compared with a reference value established forhealthy control subjects. A negative test result indicated the specimendid not contain elevated levels of lactoferrin relative to samples fromhealthy control subjects.

g. Results

One hundred forty-nine subjects having IBD were tested according to theabove procedure. Seventy-seven of the subjects, or 51.7%, were male andseventy-two of them, or 48.3%, were female. The tested male to femaleratio closely approximates the 1:1 ratio observed in the general IBDpatient population. Ages of the subjects ranged from 3 years to 78 yearsand thirty-two subjects, or 22%, were 16 years of age or younger.Seventy-seven subjects, or 51.7%, had CD and seventy-two of them, or48.3% had UC.

Thirty-one subjects having IBS were tested. Six of the subjects, or19.3%, were male and twenty-five of them, or 80.7%, were female. Thetested male to female ratio closely approximates the 1:3 ratio observedin the general IBS population. Ages of the subjects ranged from 19 yearsto 78 years.

Fifty-six healthy subjects also were tested as controls. Twenty-eight ofthe subjects, or 50%, were male and twenty-eight of them, or 50%, werefemale. Ages of the subjects ranged from infants to 79 years. A summaryof the tested subject population is illustrated in Table IX.

TABLE IX Summary of Subject Population Summary of Clinical HistoriesTotal (N = 180) Subjects Total number of IBD patients 149 No. Males 77No. Females 72 Total number of patients with CD 77 No. Males 43 No.Females 34 Total number of patients with UC 72 No. Males 34 No. Females38 Total number of patients with irritable bowel syndrome 31 No. Males 6No. Females 25 Total number of healthy persons 56 No. Males 28 No.Females 28

Fecal specimens were collected from each enrolled subject and stored at−70° C. until tested. Sample consistencies ranged from liquid to solid,numbers for which are illustrated in Table X for each subject group. Ascan be seen, forty-five of the IBD specimens were liquid specimens,sixty-two were semi-solid specimens, and forty-two were solid specimens.One of the IBS specimens was a liquid specimen, thirteen were semi-solidspecimens, and seventeen were solid specimens. All of the specimens fromhealthy control subjects were solid.

TABLE X Summary of Specimen Consistencies for Each Subject Group Summaryof Stool Specimens Total (N = 236) Specimens Total number of IBDpatients (CD and UC) 149 Total number of liquid specimens 45 Totalnumber of semi-solid specimens 62 Total number of solid specimens 42Total number of patients with IBS 31 Total number of liquid specimens 1Total number of semi-solid specimens 13 Total number of solid specimens17 Total number of healthy persons 56 Total number of liquid specimens 0Total number of semi-solid specimens 0 Total number of solid specimens56

The level of fecal lactoferrin in each specimen was determined using thequalitative lactoferrin ELISA as previously described. A specimendilution of 1:400 was used. Results were reported as positive if anoptical density of greater than or equal to 0.200 was observed.Conversely, results were reported as negative if an optical density ofless than 0.200 was observed.

Of the IBD subject group, ninety-two subjects had active disease andfifty-seven had inactive disease. Of the active group, a total of eightysubjects, or 87.0%, tested positive in the assay. Of the inactive group,a total of thirty-two subjects, or 56.1%, tested positive. Of theforty-one subjects having active UC, a total of thirty-six subjects, or87.8% tested positive in the assay. Of the fifty-one subjects havingactive CD, forty-four, or 86.3%, tested positive. All thirty-onepatients having active IBS and all fifty-six healthy control subjectstested negative in the assay. A summary of assay test results isillustrated in Table XI and various individual comparisons areillustrated in Tables XII, XIII and XIV, as more fully described below.

TABLE XI Summary of ELISA test Results for CD, UC, Active IBS, andHealthy Control Subjects Clinical Assessments N = ELISA ELISA 236 TotalPositive Negative Total IBD 149  75.2% (112) 24.8% (37) Active 92 87.0%(80) 13.0% (12) Inactive 57 56.1% (32) 43.0% (25) Total CD 77 77.9% (60)22.1% (17) Active 56 86.3% (44) 13.7% (7)  Inactive 26 61.5% (16) 38.5%(10) Total UC 72 72.2% (52) 27.7% (20) Active 41 87.8% (36) 12.2% (5) Inactive 31 51.6% (16) 48.4% (15) Total Active IBS 31 0 100.0% (31) Total Healthy Persons 56 0 100.0% (56) 

When distinguishing samples from active IBD subjects from subjectsamples having IBS or from healthy control samples, the ELISA exhibiteda sensitivity of 87% and specificity of 100%. Sensitivity was calculatedby dividing the number of persons having IBD and testing positive in theELISA by the number of subjects having IBD. Specificity was calculatedby dividing the number of subjects having IBD and testing positive inthe ELISA by the number of subjects testing positive in the ELISA. Thepredictive positive and negative values were 100% and 87.9%,respectively, and the correlation was 93.3%. These results aresummarized in Table XII.

TABLE XII Statistical Evaluation using the ELISA to Distinguish ActiveIBD from IBS/Healthy Control Subjects N = 179 Active IBD IBS/HealthyControls ELISA positive 80 0 ELISA negative 12 87 Sensitivity 87.0%Specificity 100% Predictive Positive Value 100% Predictive NegativeValue 87.9% Correlation 93.3%

When distinguishing samples from active UC subjects from subject sampleshaving IBS or from healthy control subjects, the ELISA exhibited asensitivity of 87.8% and a specificity of 100%. The predictive positiveand negative values were 100% and 94.6%, respectively, and thecorrelation was 96.1%. These results are summarized in Table XIII.

TABLE XIII Statistical Evaluation using the ELISA to Distinguish ActiveUC from IBS/Healthy Control Subjects N = 128 Active UC IBS/HealthyControls ELISA positive 36 0 ELISA negative  5 87 Sensitivity 87.8%Specificity 100% Predictive Positive Value 100% Predictive NegativeValue 94.6% Correlation 96.1%

When distinguishing subject samples having active CD from subjectsamples having IBS or from healthy control samples, the ELISA exhibiteda sensitivity of 86.3% and a specificity of 100%. The predictivepositive and negative values were 100% and 92.6%, respectively, and thecorrelation was 94.9%. These results are summarized in Table XIV.

TABLE XIV Statistical Evaluation using the ELISA to Distinguish ActiveCD from IBS/Healthy Control Subjects N = 138 Active UC IBS/HealthyControls ELISA positive 44 0 ELISA negative  7 87 Sensitivity 86.3%Specificity 100% Predictive Positive Value 100% Predictive NegativeValue 92.6% Correlation 94.9%

h. Reproducibility and Precision

The inter-assay variation was determined by analyzing eightlactoferrin-negative and eight lactoferrin-positive fecal specimens overa three day period. The average % Coefficient of Variation (CV) was23.5% for the positive specimens and 7.4% for the negative specimens.The intra-assay variation was determined by analyzing twelve fecalspecimens using six replicates in one lot of kits. The intra-assayanalysis ranged in % CV from 2.7 to 24.0 with an average of 8.7%.

2. Quantitative Assay

In the quantitative assay of the present invention, fecal specimenspreferably are serially diluted ten-fold and added to microtiter wellscontaining immobilized polyclonal antibodies against human lactoferrin.If endogenous lactoferrin is present, it will bind to the antibodiesduring an incubation at approximately 37° C. Following the incubation,conjugate comprised of polyclonal antibodies coupled to horseradishperoxidase enzyme is added and allowed to bind to captured lactoferrin.Unbound conjugate is then washed from the well and a component substrate(e.g., tetra-methyl-benzidene and hydrogen peroxide) is added for colordevelopment. Following the substrate incubation, 0.6N sulfuric acid isadded to quench the reaction and the absorbance or optical density (OD)is obtained spectrophotometrically at 450 nm on a single wavelengthdevice. Fecal lactoferrin concentrations are determined by comparison toa standard curve generated using purified human lactoferrin.

a. Preparation of Standard Curve

A 1 mg/mL stock solution of purified human lactoferrin, manufactured bySigma Immunochemicals of St. Louis, Mo., was prepared using 10 mg oflactoferrin dissolved in 10 mL of sterile phosphate buffered saline(PBS) at a pH of 7.4. Serial two-fold dilutions of lactoferrin were madeusing the range of approximately 6 to 100 ng/mL in Diluent. For theanalysis, 0.1 mL of each standard was assayed in duplicate. Opticaldensities (OD₄₅₀) were determined and plotted versus lactoferrinconcentration to generate standard curves. The linear portion of thecurve was determined by linear regression analysis using the Log-Logmethod (Microsoft EXCEL, Microsoft R Office). The lowest dilution ofspecimen that gave an OD₄₅₀ within the linear portion of the curve wasused to determine the lactoferrin concentration. The final concentrationwas obtained by multiplying the concentration by the dilution factor.

b. Quantitative Test Procedure

In order to assess the ability of the quantitative ELISA to measure thelevel of fecal lactoferrin, two fecal specimens collected six weeksapart from six female and five male adults were diluted and then spikedwith lactoferrin to a concentration of 25 ng/mL. The “EstimatedLactoferrin” that was determined represents the level of lactoferrindetermined from a standard curve generated with the quantitative ELISA.The % Variation represents the difference between the “Actual” amountused to spike the sample and the “Estimated” amount. Under theseconditions, the variations ranged from 1.0% to 85.8% for females and8.8% to 47.0% for males. Results showed a higher percent variation infemale adults as compared to male adults. The stool samples that showeda higher variation had higher levels of lactoferrin prior to spiking.The results are illustrated in Tables XV and XVI below.

TABLE XV Stool samples of female adult subjects spiked to a finalconcentration of 25 ng/mL Actual Estimated Patient LactoferrinLactoferrin ID # (ng/ML) (ng/mL) Variation (%) 1 25 15.4 38.4 2 25 22.98.5 3 25 21.8 12.7 4 25 28.4 13.5 5 25 16.2 35.3 6 25 15.8 37.0 7 2535.5 41.8 8 25 46.5 85.8 9 25 27.7 10.8 10 25 32.3 29.1 11 25 26.1 4.312 25 25.3 1.0

TABLE XVI Stool samples of male adult subjects spiked to a finalconcentration of 25 ng/mL Actual Estimated Patient LactoferrinLactoferrin ID # (ng/ML) (ng/mL) Variation (%) 1 25 21.9 12.4 2 25 21.215.0 3 25 20.9 16.3 4 25 21.4 14.4 5 25 20.8 16.8 6 25 22.8 8.8 7 2528.9 15.5 8 25 29.4 17.4 9 25 36.7 47.0 10 25 19.5 21.9

A second method for spiking was using the same two stool specimenscollected six weeks apart from six female and five male adults werediluted and spiked with lactoferrin to a concentration of 4 μg/mL. The“Estimated Lactoferrin” represents the level of lactoferrin determinedfrom a standard curve generated by the quantitative ELISA. The %Variation represents the difference between the “Actual” amount used tospike the sample and the “Estimated” value. Under these conditions, thevariation ranged from 11.3% to 84.9% for females and from 5.0% to 39.2%for males. Results were similar to those obtained with specimens spikedwith 25 ng/mL lactoferrin as described above, showing a higher percentvariation in female adults compared to male adults. The results areillustrated in Tables XVII and XVIII below.

TABLE XVII Stool samples of female adult subjects spiked to a finalconcentration of 4 μg/mL Actual Estimated Patient LactoferrinLactoferrin ID # (μg/mL) (μg/mL) Variation (%) 1 4 4.5 11.3 2 4 4.6 15.33 4 5.3 33.4 4 4 4.9 21.4 5 4 3.5 11.5 6 4 3.4 14.7 7 4 5.3 32.7 8 4 6.767.6 9 4 5.5 38.6 10 4 5.8 44.9 11 4 5.8 43.9 12 4 7.4 84.9

TABLE XVIII Stool samples of male adult subjects spiked to a finalconcentration of 4 μg/mL Actual Estimated Patient LactoferrinLactoferrin ID # (μg/mL) (μg/mL) Variation (%) 1 4 4.7 17.5 2 4 4.6 14.43 4 4.2 5.0 4 4 5.6 39.2 5 4 4.2 5.9 6 4 4.7 18.5 7 4 4.7 16.5 8 4 5.537.9 9 4 5.3 33.6 10 4 4.3 6.6

3. Monitoring Using the Quantitative ELISA

The quantitative ELISA of the present invention was used to follow thelactoferrin levels of single patient suffering from ulcerative colitisduring a “flare” of active disease through remission. The patient showedextremely high levels of lactoferrin (e.g., 9749.37 μg/mL feces) duringthe peak of the active disease, the levels dropping rapidly (e.g., to7.42 μg/mL feces) following anti-inflammatory drug therapy. Levelselevated dramatically again during a relapse and leveled at slightlyabove those of healthy control persons (e.g., 11.06 μg/mL feces) duringperiods of remission. Thus, lactoferrin levels determined according tothe quantitative ELISA of the present invention accurately depicteddisease activity in response to medical treatment.

In summary, the present invention is directed to non-invasive methodsfor differentiating between irritable bowel syndrome and inflammatorybowel disease using the presence of fecal lactoferrin as a detectionmarker for fecal leukocytes, and a kit used for such method. The presentinvention is further directed to immunoassays that utilize antibodiesspecific to human lactoferrin for the measurement of total endogenouslactoferrin in human feces. Still further, the present invention isdirected to a quantitative immunoassay for monitoring the levels offecal lactoferrin in a patient having IBD.

The immunoassays of the present invention are sensitive, specific andeasy to perform. The assays detect lactoferrin, a stable protein thatserves as a detection marker for fecal leukocytes and an indicator ofintestinal inflammation, and quantitate fecal lactoferrin levels formonitoring patients having IBD. The tests are rapid and can be completedwithin about seventy-five minutes. Research results support the use ofthe qualitative ELISA as an in vitro diagnostic aid to help distinguishactive IBD patients from those with active IBS. Research results furthersupport the use of the quantitative ELISA for monitoring levels of fecallactoferrin in patients having inflammatory diseases. The presentinvention has been described in relation to particular embodiments whichare intended in all respects to be illustrative rather than restrictive.Alternative embodiments will become apparent to those skilled in the artto which the present invention pertains without departing from itsscope.

From the foregoing, it will be seen that this invention is one welladapted to attain all the ends and objects herein above set forthtogether with other advantages which are obvious and which are inherentto the method. It will be understood that certain features andsubcombinations are of utility and may be employed without reference toother features and subcombinations. This is contemplated by and iswithin the scope of the claims.

1. A method for diagnosing irritable bowel syndrome, the methodcomprising: obtaining a fecal sample from a person presenting withsymptoms common to inflammatory bowel disease and irritable bowelsyndrome; diluting the sample; measuring the sample to determine thesample does not contain an elevated level of endogenous lactoferrin; andupon determining the sample does not contain an elevated level ofendogenous lactoferrin, diagnosing the person with irritable bowelsyndrome.
 2. The method of claim 1, wherein the step of diluting saidsample comprises diluting said sample to a 1:400 dilution factor.
 3. Themethod of claim 1, wherein the step of measuring the sample to determinethe sample does not contain an elevated level of endogenous lactoferrincomprises contacting the sample with immobilized polyclonal antibodiesto human lactoferrin to create an antibody bound sample.
 4. The methodof claim 3, wherein the step of measuring the sample to determine thesample does not contain an elevated level of endogenous lactoferrincomprises contacting the antibody bound sample with enzyme-linkedpolyclonal antibodies such that the enzyme-linked polyclonal antibodiesare allowed to bind to capture lactoferrin and create an enzyme-linkedantibody bound sample.
 5. The method of claim 4, wherein the step ofmeasuring the sample to determine the sample does not contain anelevated level of endogenous lactoferrin comprises adding a substrate tothe enzyme-linked antibody bound sample for color development to createa readable enzyme-linked antibody bound sample.
 6. The method of claim5, wherein the step of measuring the sample to determine the sample doesnot contain an elevated level of endogenous lactoferrin comprisesdetermining an optical density of said readable enzyme-linked antibodybound sample at 450 nm, wherein the optical destiny corresponds to alevel of endogenous lactoferrin in the readable enzyme-linked antibodybound sample.
 7. The method of claim 6, wherein if said optical densityof said readable enzyme-linked antibody bound sample is greater than0.200, said sample contains an elevated level of endogenous lactoferrin.8. A method for monitoring a human having inflammatory bowel disease forgastrointestinal inflammation, the method comprising: obtaining a firstfecal sample from a human having inflammatory bowel disease at a firsttime; determining the concentration of endogenous lactoferrin in saidfirst fecal sample to obtain a first lactoferrin concentration;obtaining a second fecal sample from the human having inflammatory boweldisease at a time later than the first time; determining theconcentration of endogenous lactoferrin in said second fecal sample toobtain a second lactoferrin concentration; and comparing said firstlactoferrin concentration to said second lactoferrin concentration todetermine whether the human has had an increase or decrease ingastrointestinal inflammation.
 9. The method of claim 8, furthercomprising: diluting said first and second samples.
 10. The method ofclaim 9, further comprising: contacting said first sample withimmobilized polyclonal antibodies to endogenous lactoferrin to create afirst treated sample; contacting said first treated sample withenzyme-linked polyclonal antibodies to create a first enzyme-linkedantibody bound sample; adding a substrate to the first enzyme-linkedantibody bound sample to create a first readable sample; and determiningthe optical density of said first readable sample.
 11. The method ofclaim 10, further comprising: contacting said second sample withimmobilized polyclonal antibodies to endogenous lactoferrin to create asecond treated sample; contacting said second treated sample withenzyme-linked polyclonal antibodies to create a second enzyme-linkedantibody bound sample; adding a substrate to the second enzyme-linkedantibody bound sample to create a second readable sample; anddetermining the optical density of said second readable sample.
 12. Themethod of claim 10, further comprising: generating a purifiedlactoferrin standard curve and determining a linear portion of thestandard curve.
 13. The method of claim 12, wherein the step of dilutingsaid fecal sample comprises diluting said first and second samples byserial tenfold dilutions until a measured result indicates aconcentration of fecal lactoferrin that provides an optical densityreading at 450 nm that is within the linear portion of the standardcurve.
 14. The method of claim 8, further comprising: monitoring thehuman for changing levels of fecal lactoferrin as an indicator ofeffectiveness of medical therapy.